1. Lipid Biosynthesis BISC 6310 Dr. Fayez Almabhouh Advanced Biochemistry
Advanced Physiology BISC Dr. Fayez Almabhouh

2. Introduction
Lipids play a variety of cellular roles.
They are the principal form of stored energy in most organisms and major constituents of cellular membranes.

3. Introduction Specialized lipids serve as: Pigments (retinal, carotene)
Cofactors (vitamin K)
Detergents (bile salts)
Transporters (dolichols)
Hormones (vitamin D derivatives, sex hormones)
Extracellular and intracellular messengers (eicosanoids, phosphatidylinositol derivatives)
Anchors for membrane proteins (covalently attached fatty acids, and phosphatidylinositol).

4. Introduction
The ability to synthesize a variety of lipids is essential to all organisms.
Like other biosynthetic pathways, these reaction sequences are endergonic and reductive.
They use ATP as a source of metabolic energy and a reduced electron carrier (usually NADPH) as reductant.

5. Biosynthesis of Fatty Acids
Fatty acid oxidation takes place by the oxidative removal of successive two-carbon (acetyl-CoA) units, biochemists thought the biosynthesis of fatty acids might proceed by a simple reversal of the same enzymatic steps.

6. However, as they were to find out, fatty acid biosynthesis and breakdown occur by different pathways, are catalyzed by different sets of enzymes, and take place in different parts of the cell.
Moreover, biosynthesis requires the participation of a three-carbon intermediate, Malonyl-CoA, that is not involved in fatty acid breakdown.

7. Malonyl-CoA Is Formed from Acetyl-CoA and Bicarbonate
The formation of malonyl-CoA from acetyl-CoA is an irreversible process, catalyzed by acetyl-CoA carboxylase.
The enzyme contains a biotin prosthetic group covalently bound in amide linkage to the ε-amino group of a Lys residue in one of the three polypeptides or domains of the enzyme molecule.

8. The two-step reaction catalyzed by this enzyme is very similar to other biotin-dependent carboxylation reactions, such as those catalyzed by pyruvate carboxylase and propionyl-CoA carboxylase.

9. The carboxyl group, derived from bicarbonate (HCO3-), is first transferred to biotin in an ATP dependent reaction.

10. The biotinyl group serves as a temporary carrier of CO2, transferring it to acetyl-CoA in the second step to yield malonyl-CoA.

12. Fatty acid synthesis: proceeds in a repeating reaction sequence.
The long carbon chains of fatty acids are assembled in a repeating four-step sequence.

13. The Mammalian Fatty Acid Synthase Has Multiple Active Sites
There are two major variants of fatty acid synthase: fatty acid synthase I (FAS I), found in vertebrates and fungi, and fatty acid synthase II (FAS II), found in plants and bacteria.
The FAS I found in vertebrates consists of a single multifunctional polypeptide chain
The mammalian FAS I is the prototype.
Seven active sites for different reactions lie in separate domains

14. FAS II, in plants and bacteria, is a dissociated system; each step in the synthesis is catalyzed by a separate and freely diffusible enzyme.
Intermediates are also diffusible and may be diverted into other pathways

15. All of the active sites in the mammalian system are located in different domains within a single large polypeptide chain. The different enzymatic activites are:
β-ketoacyl-ACP synthase (KS)
Malonyl/acetyl-CoA–ACP transferase(MAT)
β -hydroxyacyl-ACP dehydratase (DH)
Enoyl-ACP reductase (ER)
β -ketoacyl-ACP reductase (KR).
ACP is the acyl carrier protein.
The seventh domain (TE) is a thioesterase that releases the palmitate product from ACP when the synthesis is completed.

16. Before the condensation reactions that build up the fatty acid chain can begin, the two thiol groups on the enzyme complex must be charged with the correct acyl groups.

17. Fatty Acid Synthase Receives the Acetyl and Malonyl Groups
First, the acetyl group of acetyl- CoA is transferred to ACP in a reaction catalyzed by the malonyl/ acetyl-CoA–ACP transferase (MAT) domain of the multifunctional polypeptide.
The acetyl group is then transferred to the Cys —SH group of the β -ketoacyl-ACP synthase (KS)

18. The second reaction, transfer of the malonyl group from malonyl-CoA to the -SH group of ACP, is catalyzed by MAT.

19. A four-step sequence:
Step 1. Condensation: The first reaction in the formation of a fatty acid chain is condensation of the activated acetyl and malonyl groups
to form acetoacetyl-ACP, an acetoacetyl group bound to ACP through the phosphopantetheine -SH group; simultaneously, a molecule of CO2 is produced.

20. In this reaction, catalyzed by β-ketoacyl-ACP synthase (KS), the acetyl group is transferred from the Cys -SH group of the enzyme to the malonyl group on the -SH of ACP, becoming the methyl-terminal two-carbon unit of the new acetoacetyl group.
The carbon atom of the CO2 formed in this reaction is the same carbon originally introduced into malonyl- CoA from HCO3 by the acetyl-CoA carboxylase reaction . Thus CO2 is only transiently in covalent linkage during fatty acid biosynthesis; it is removed as each two-carbon unit is added.

21. Step 2. Reduction of the Carbonyl Group The acetoacetyl- ACP formed in the condensation step now undergoes reduction of the carbonyl group at C-3 to form D-β-hydroxybutyryl-ACP. This reaction is catalyzed by β - ketoacyl-ACP reductase (KR) and the electron donor is NADPH.

22. Step 3. Dehydration The elements of water are now removed from C-2 and C-3 of D-β-hydroxybutyryl-ACP to yield a double bond in the product, trans-Δ2- butenoyl-ACP. The enzyme that catalyzes this dehydration is β - hydroxyacyl-ACP dehydratase (HD).

23. Step 4. Reduction of the Double Bond : Finally, the double bond of trans-Δ2-butenoyl-ACP is reduced (saturated) to form butyryl-ACP by the action of enoyl-ACP reductase (ER); again, NADPH is the electron donor.

24. A saturated acyl group produced by this set of reactions becomes the substrate for subsequent condensation with an activated malonyl group.

25. Translocation of butyryl group to Cys on β-ketoacyl-ACP synthase (KS)

26. Recharging of ACP with another malonyl group (MAT)
FIGURE 21-6 (part 8) Sequence of events during synthesis of a fatty acid. The mammalian FAS I complex is shown schematically, with catalytic domains colored as in Figure Each domain of the larger polypeptide represents one of the six enzymatic activities of the complex, arranged in a large, tight "S" shape. The acyl carrier protein (ACP) is not resolved in the crystal structure shown in Figure 21-3, but is attached to the KS domain. The phosphopantetheine arm of ACP ends in an —SH. After the first panel, the enzyme shown in color is the one that will act in the next step. As in Figure 21-4, the initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. Steps 1 to 4 are described in the text.

27. Beginning of the second round of the fatty acid synthesis cycle.
The butyryl group is on the Cys -SH group. The incoming malonyl group is first attached to the phosphopantetheine -SH group.
Then, in the condensation step, the entire butyryl group on the Cys -SH is exchanged for the carboxyl group of the malonyl residue, which is lost as CO2 (green). This step is analogous to step 1.
The product, a six-carbon β-ketoacyl-ACP, now contains four carbons derived from malonyl-CoA and two derived from the acetyl-CoA that started the reaction. The β-ketoacyl group then undergoes steps 2 through 4

29. With each passage through the cycle, the fatty acyl chain is extended by two carbons.
When the chain length reaches 16 carbons, the product (palmitate, 16:0) leaves the cycle.

30. The Fatty Acid Synthase Reactions Are Repeated to Form Palmitate
The overall process of palmitate synthesis. The fatty acyl chain grows by two-carbon units donated by activated malonate, with loss of CO2 at each step. The initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. After each two-carbon addition, reductions convert the growing chain to a saturated fatty acid of four, then six, then eight carbons, and so on. The final product is palmitate (16:0).

31. Seven cycles of condensation and reduction produce the 16-carbon saturated palmitoyl group, still bound to ACP.
For reasons not well understood, chain elongation by the synthase complex generally stops at this point and free palmitate is released from the ACP by a hydrolytic activity (thioesterase; TE) in the multifunctional protein.

33. Fatty Acid Synthesis Occurs in the Cytosol of Many Organisms but in the Chloroplasts of Plants
In most higher eukaryotes, the fatty acid synthase complex is found exclusively in the cytosol, as are the biosynthetic enzymes for nucleotides, amino acids, and glucose.

34. Shuttle for transfer of acetyl groups from mitochondria to the cytosol.

35. Regulation of fatty acid synthesis

36. Long-Chain Saturated Fatty Acids Are Synthesized from Palmitate
Palmitate, the principal product of the fatty acid synthase system in animal cells, is the precursor of other long-chain fatty acids.
It may be lengthened to form stearate (18:0) or even longer saturated fatty acids by further additions of acetyl groups, through the action of fatty acid elongation systems present in the smooth endoplasmic reticulum and in mitochondria

37. Routes of synthesis of other fatty acids
Routes of synthesis of other fatty acids. Palmitate is the precursor of stearate and longer-chain saturated fatty acids, as well as the monounsaturated acids palmitoleate and oleate.

38. Biosynthesis of Triacylglycerols
Most of the fatty acids synthesized or ingested by an organism have one of two fates: incorporation into triacylglycerols for the storage of metabolic energy or incorporation into the phospholipid components of membranes.

39. The partitioning between these alternative fates depends on the organism’s current needs.
During rapid growth, synthesis of new membranes requires the production of membrane phospholipids; when an organism has a plentiful food supply but is not actively growing, it shunts most of its fatty acids into storage fats.
Both pathways begin at the same point: the formation of fatty acyl esters of glycerol.

40. Animals can synthesize and store large quantities of triacylglycerols, to be used later as fuel.
Humans can store only a few hundred grams of glycogen in liver and muscle, barely enough to supply the body’s energy needs for 12 hours.

41. In contrast, the total amount of stored triacylglycerol in a 70-kg man of average build is about 15 kg, enough to support basal energy needs for as long as 12 weeks.

42. Triacylglycerols have the highest energy content of all stored nutrients—more than 38 kJ/g.
Whenever carbohydrate is ingested in excess of the organism’s capacity to store glycogen, the excess is converted to triacylglycerols and stored in adipose tissue.
Plants also manufacture triacylglycerols as an energy-rich fuel, mainly stored in fruits, nuts, and seeds.

43. Triacylglycerols and Glycerophospholipids are synthesized from the same precursors
In animal tissues, triacylglycerols and glycerophospholipids such as phosphatidylethanolamine share two precursors (fatty acyl–CoA and L-glycerol 3-phosphate) and several biosynthetic steps.

44. The vast majority of the glycerol 3-phosphate is derived from the glycolytic intermediate dihydroxyacetone phosphate (DHAP) by the action of the cytosolic NAD-linked glycerol 3-phosphate dehydrogenase; in liver and kidney, a small amount of glycerol 3-phosphate is also formed from glycerol by the action of glycerol kinase (Fig).

45. Glycerol 3-phosphate
formation

46. The other precursors of triacylglycerols are fatty acyl–CoAs, formed from fatty acids by acyl-CoA synthetases, the same enzymes responsible for the activation of fatty acids for β-oxidation

47. The first stage in the biosynthesis of triacylglycerols is the acylation of the two free hydroxyl groups of L-glycerol 3-phosphate by two molecules of fatty acyl–CoA to yield diacylglycerol 3-phosphate, more commonly called phosphatidic acid or phosphatidate
diacylglycerol 3-phosphate

48. Phosphatidic acid is present in only trace amounts in cells but is a central intermediate in lipid biosynthesis; it can be converted either to a triacylglycerol or to a glycerophospholipid.

49. In the pathway to triacylglycerols, phosphatidic acid is hydrolyzed by phosphatidic acid phosphatase to form a 1,2-diacylglycerol.
Diacylglycerols are then converted to triacylglycerols by transesterification with a third fatty acyl–CoA.

50. Triacylglycerol biosynthesis in animals is regulated by hormones
The rate of triacylglycerol biosynthesis is profoundly altered by the action of several hormones.
Insulin, for example, promotes the conversion of carbohydrate to triacylglycerols.
People with severe diabetes mellitus, due to failure of insulin secretion or action, not only are unable to use glucose properly but also fail to synthesize fatty acids from carbohydrates or amino acids.

51. Regulation of triacylglycerol synthesis by insulin
Regulation of triacylglycerol synthesis by insulin. Insulin stimulates conversion of dietary carbohydrates and proteins to fat. Individuals with diabetes mellitus lack insulin; in uncontrolled disease, this results in diminished fatty acid synthesis, and the acetyl-CoA arising from catabolism of carbohydrates and proteins is shunted instead to ketone body production and therefore lose weight. .

52. An additional factor in the balance between biosynthesis and degradation of triacylglycerols is that approximately 75% of all fatty acids released by lipolysis are reesterified to form triacylglycerols rather than used for fuel.
This ratio persists even under starvation conditions, when energy metabolism is shunted from the use of carbohydrate to the oxidation of fatty acids.

53. Some of this fatty acid recycling takes place in adipose tissue, with the reesterification occurring before release into the bloodstream.
Some takes place via a systemic cycle in which free fatty acids are transported to liver, recycled to triacylglycerol, exported again to the blood, and taken up again by adipose tissue after release from triacylglycerol by extracellular lipoprotein lipase.

54. Triacylglycerol cycle
Triacylglycerol cycle. In mammals, triacylglycerol molecules are broken down and resynthesized in a triacylglycerol cycle during starvation.

55. When the mobilization of fatty acids is required to meet energy needs, release from adipose tissue is stimulated by the hormones glucagon and epinephrine.
Simultaneously, these hormonal signals decrease the rate of glycolysis and increase the rate of gluconeogenesis in the liver (providing glucose for the brain).

56. Adipose Tissue Generates Glycerol 3-Phosphate by Glyceroneogenesis
The pathway is essentially an abbreviated version of gluconeogenesis, from pyruvate to dihydroxyacetone phosphate (DHAP), followed by conversion of DHAP to glycerol 3-phosphate, which is used for the synthesis of triacylglycerol

57. Regulation of glyceroneogenesis
Regulation of glyceroneogenesis. (a) Glucocorticoid hormones stimulate glyceroneogenesis and gluconeogenesis in the liver, while suppressing glyceroneogenesis in the adipose tissue (by reciprocal regulation of the gene expressing PEP carboxykinase (PEPCK) in the two tissues); this increases the flux through the triacylglycerol cycle. The glycerol freed by the breakdown of triacylglycerol in adipose tissue is released to the blood and transported to the liver, where it is primarily converted to glucose, although some is converted to glycerol 3-phosphate by glycerol kinase.

58. (b) A class of drugs called thiazolidinediones are now used to treat type 2 diabetes. In this disease, high levels of free fatty acids in the blood interfere with glucose utilization in muscle and promote insulin resistance. Thiazolidinediones activate a nuclear receptor called peroxisome proliferator-activated receptor γ (PPAR γ ), which induces the activity of PEP carboxykinase. Therapeutically, thiazolidinediones increase the rate of glyceroneogenesis, thus increasing the resynthesis of triacylglycerol in adipose tissue and reducing the amount of free fatty acid in the blood.

59. Biosynthesis of membrane phospholipids
All the biosynthetic pathways follow a few basic patterns. In general, the assembly of phospholipids from simple precursors requires:
(1) Synthesis of the backbone molecule (glycerol or
sphingosine).
(2) Attachment of fatty acid(s) to the backbone through an ester or amide linkage.
(3) Addition of a hydrophilic head group to the backbone through a phosphodiesterlinkage; and, in some cases,
(4) Alteration or exchange of the head group to yield the final phospholipid product.

60. In eukaryotic cells, phospholipid synthesis occurs primarily on the surfaces of the smooth endoplasmic reticulum and the mitochondrial inner membrane.
Some newly formed phospholipids remain at the site of synthesis, but most are destined for other cellular locations.

61. Cells have two strategies for attaching phospholipid head groups
The first steps of glycerophospholipid synthesis are shared with the pathway to triacylglycerols : two fatty acyl groups are esterified to C-1 and C-2 of L-glycerol 3-phosphate to form phosphatidic acid.
Commonly but not invariably, the fatty acid at C-1 is saturated and that at C-2 is unsaturated.
A second route to phosphatidic acid is the phosphorylation of a diacylglycerol by a specific kinase.

62. Head-group attachment
Head-group attachment. The phospholipid head group is attached to a diacylglycerol by a phosphodiester bond, formed when phosphoric acid condenses with two alcohols, eliminating two molecules of H2O.

63. In the biosynthetic process, one of the hydroxyls is first activated by attachment of a nucleotide, cytidine diphosphate (CDP).
Cytidine monophosphate (CMP) is then displaced in a nucleophilic attack by the other hydroxyl.

64. The CDP is attached either to the diacylglycerol, forming the activated phosphatidic acid CDP-diacylglycerol (strategy 1), or to the hydroxyl of the head group (strategy 2).

65. Eukaryotic cells use both strategies
(occurring on sER and
inner membrane of
mitochondria)
Bacteria mainly use
this strategy

66. Origin of the polar head groups of phospholipids in E. coli
Origin of the polar head groups of phospholipids in E. coli. Initially, a head group (either serine or glycerol 3-phosphate) is attached via a CDP diacylglycerol intermediate (strategy 1

67. Eukaryotic pathways to phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine are interrelated.
Yeast, like bacteria, can produce phosphatidylserine by condensation of CDP-diacylglycerol and serine, and can synthesize phosphatidylethanolamine from phosphatidylserine in the reaction catalyzed by phosphatidylserine decarboxylase

68. Phosphatidylethanolamine may also be converted to phosphatidylcholine (lecithin) by the addition of three methyl groups to its amino group; Sadenosylmethionine is the methyl group donor for all three methylation reactions.
adoMet is S-adenosylmethionine;
adoHcy, Sadenosylhomocysteine.

69. In mammals, phosphatidylserine is not synthesized from CDP-diacylglycerol; instead, it is derived from phosphatidylethanolamine via the head-group exchange reaction.

70. Pathway for phosphatidylcholine synthesis from choline in mammals
Pathway for phosphatidylcholine synthesis from choline in mammals. The same strategy shown here (strategy 2) is also used for salvaging ethanolamine in phosphatidylethanolamine synthesis.

71. Biosynthesis of Cholesterol
Cholesterol is doubtless the most publicized lipid, notorious because of the strong correlation between high levels of cholesterol in the blood and the incidence of human cardiovascular diseases.
Less well advertised is cholesterol’s crucial role as a component of cellular membranes and as a precursor of steroid hormones and bile acids.
Cholesterol is an essential molecule in many animals, including humans, but is not required in the mammalian diet—all cells can synthesize it from simple precursors.

72. Cholesterol Is Made from Acetyl-CoA in Four Stages
Cholesterol, like long-chain fatty acids, is made from acetyl-CoA, but the assembly plan is quite different.
In early experiments, animals were fed acetate labeled with 14C in either the methyl carbon or the carboxyl carbon.
The pattern of labeling in the cholesterol isolated from the two groups of animals provided the blueprint for working out the enzymatic steps in cholesterol biosynthesis.
Figure: Origin of the carbon atoms of cholesterol. This can be deduced from tracer experiments with acetate labeled in the methyl carbon (black) or the carboxyl carbon (red). The individual rings in the fused-ring system are designated A through D.

73. Summary of the four stages of cholesterol biosynthesis.

74. Stage 1: Synthesis of Mevalonate from Acetate
The first stage in cholesterol biosynthesis leads to the intermediate mevalonate.
Two molecules of acetyl-CoA condense to form acetoacetyl-CoA, which condenses with a third molecule of acetyl-CoA to yield the six-carbon compound HMG-CoA. These first two reactions are catalyzed by thiolase and HMG-CoA synthase.
The third reaction is the committed and rate-limiting step: reduction of HMG-CoA to mevalonate, for which each of two molecules of NADPH donates two electrons

75. Stage 2 Conversion of Mevalonate to Two Activated Isoprenes:
In the next stage of cholesterol synthesis, three phosphate groups are transferred from three ATP molecules to mevalonate.
The phosphate attached to the C-3 hydroxyl group of mevalonate and the nearby carboxyl group removed.
This is the first of the two activated isoprenes central to cholesterol formation.
Δ3-isopentenyl pyrophosphate and
dimethylallyl pyrophosphate

76. Stage 3 condensation of six activated isoprene units to form squalene
Formation of squalene. This 30-carbon structure arises through successive condensations of activated isoprene (five-carbon) units.

77. stage 4 Conversion of squalene to the four-ring steroid nucleus
Ring closure converts linear squalene to the condensed steroid nucleus. The first step in this sequence is catalyzed by a mixed-function oxidase (a monooxygenase), for which the cosubstrate is NADPH. The product is an epoxide.

78. The epoxide then cyclized to the steroid nucleus
The epoxide then cyclized to the steroid nucleus. The final product of these reactions in animal cells is cholesterol; in other organisms, slightly different sterols are produced.

79. In animal cells, this cyclization results in the formation of lanosterol, which contains the four rings characteristic of the steroid nucleus. Lanosterol is finally converted to cholesterol in a series of about 20 reactions that include the migration of some methyl groups and the removal of others.

80. Elucidation of this extraordinary biosynthetic pathway, one of the most complex known, was accomplished by Konrad Bloch, Feodor Lynen, John Cornforth, and George Popják in the late 1950s

81. Cholesterol is the sterol characteristic of animal cells
Plants, fungi, and protists make other, closely related sterols instead.
They use the same synthetic pathway as far as squalene 2,3-epoxide, at which point the pathways diverge slightly, yielding other sterols, such as stigmasterol in many plants and ergosterol in fungi

83. Cholesterol Has Several Fates
Much of the cholesterol synthesis in vertebrates takes place in the liver. A small fraction of the cholesterol made there is incorporated into the membranes of hepatocytes, but most of it is exported in one of three forms: biliary cholesterol, bile acids, or cholesteryl esters.
Bile acids and their salts are relatively hydrophilic cholesterol derivatives that are synthesized in the liver and aid in lipid digestion

84. Cholesteryl esters are formed in the liver through the action of acyl-CoA–cholesterol acyl transferase (ACAT). This enzyme catalyzes the transfer of a fatty acid from coenzyme A to the hydroxyl group of cholesterol converting the cholesterol to a more hydrophobic form.
Cholesteryl esters are transported in secreted lipoprotein particles to other tissues that use cholesterol, or they are stored in the liver

85. Lipoproteins and lipid transport

86. All growing animal tissues need cholesterol for membrane synthesis, and some organs (adrenal gland and gonads, for example) use cholesterol as a precursor for steroid hormone production.
Cholesterol is also a precursor of vitamin D.